Technical Abstract: Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources of selenomethionine, the purified L-isomer (SeMet) and an in vitro-digested, Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC). Interestingly, SeMSC, SeMet and digested Se-enriched yeast were transported at comparable rates from the apical to basolateral sides, each being about 3 fold that of selenite. This observation suggests the presence of a transcellular pathway for Se-amino acids. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, had comparable potential to support glutathione peroxidase (GPx) activity. Because selenoprotein P has been postulated to be a key Se transport protein, we also examined its expression and found that the addition of selenite, SeMSC, SeMet or digested Se-enriched yeast did not change intracellular selenoprotein P expression in cells cultured in serum-contained media. Taken together, these studies show that the chemical form of Se can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.